Aspergillus Sydowii and Other Potential Fungal Pathogens in Gorgonian Octocorals of the Ecuadorian Pacific

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RESEARCH ARTICLE Aspergillus sydowii and Other Potential Fungal Pathogens in Gorgonian Octocorals of the Ecuadorian Pacific

M. Mar Soler-Hurtado1,2, JoseÂ Vladimir Sandoval-Sierra3, Annie Machordom1, Javier DieÂguez-Uribeondo3*

1 Departamento de Biodiversidad y BiologõÂa Evolutiva, Museo Nacional de Ciencias Naturales (MNCN- CSIC), Madrid, Spain, 2 Departamento de Biodiversidad y EcologõÂa de Invertebrados Marinos, Facultad de BiologõÂa, Universidad de Sevilla, Sevilla, Spain, 3 Departamento de MicologõÂa, Real JardõÂn BotaÂnico CSIC, a11111 Madrid, Spain * [email protected]

Abstract

Emerging fungal diseases are threatening ecosystems and have increased in recent OPEN ACCESS decades. In corals, the prevalence and consequences of these infections have also Citation: Soler-Hurtado MM, Sandoval-Sierra JV, increased in frequency and severity. Coral reefs are affected by an emerging fungal disease Machordom A, Die´guez-Uribeondo J (2016) Aspergillus sydowii and Other Potential Fungal named aspergillosis, caused by Aspergillus sydowii. This disease and its pathogen have Pathogens in Gorgonian Octocorals of the been reported along the Caribbean and Pacific coasts of Colombia. Despite this, an impor- Ecuadorian Pacific. PLoS ONE 11(11): e0165992. tant number of coral reefs worldwide have not been investigated for the presence of this doi:10.1371/journal.pone.0165992 pathogen. In this work, we carried out the surveillance of the main coral reef of the Ecuador- Editor: Kenneth So¨derha¨ll, Uppsala University, ian Pacific with a focus on the two most abundant and cosmopolitan species of this ecosys- SWEDEN tem, Leptogorgia sp. and Leptogorgia obscura. We collected 59 isolates and obtained the Received: September 20, 2016 corresponding sequences of the Internal Transcribed Spacers (ITS) of the ribosomal DNA. Accepted: October 22, 2016 These were phylogenetically analyzed using MrBayes, which indicated the presence of two

Published: November 30, 2016 isolates of the coral reef pathogen A. sydowii, as well as 16 additional species that are potentially pathogenic to corals. Although the analyzed gorgonian specimens appeared Copyright: © 2016 Soler-Hurtado et al. This is an open access article distributed under the terms of healthy, the presence of these pathogens, especially of A. sydowii, alert us to the potential the Creative Commons Attribution License, which risk to the health and future survival of the Pacific Ecuadorian coral ecosystem under the permits unrestricted use, distribution, and current scenario of increasing threats and stressors to coral reefs, such as habitat alter- reproduction in any medium, provided the original ations by humans and global climate change. author and source are credited.

Data Availability Statement: Cultures were labeled as ASP001 through ASP059 in the culture collection of the Real Jardı´n Bota´nico, Madrid, Spain. The molecular data are published in GenBank (GenBank number are included in the Introduction manuscript). Coral reefs are considered one of the most biologically diverse ecosystems in the marine realm Funding: This research was only partially [1]. They maintain a high biomass and abundance of varied organisms [2] and provide a pleth- supported by a grant from the Spanish Ministry of ora of micro-habitats to support enormous biodiversity [3–6]. In recent decades, coral reefs Economy and Competitiveness (CTM2014-57949- R). The authors have not had any additional have experienced increasing pressures, and are disturbed by a combination of direct human funding and coauthors have supported the impacts, e.g., habitat fragmentation and reduction of functional diversity [7], and global cli- sampling and sequencing from other projects. mate change, e.g., increasing ocean acidification and temperature, coral bleaching, etc. [8].

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Both author and coauthors have currently not These conditions make reefs more susceptible to the proliferation and development of oppor- funding to pay publication fees. tunistic organisms, which take advantage of the weakened corals [9,10]. Competing Interests: The authors have declared The coral disease aspergillosis has produced significant deterioration and partial and mas- that no competing interests exist. sive mortalities of coral communities in the Caribbean Sea [11–15]. The responsible pathogen is the ascomycetous fungus Aspergillus sydowii (Bainier and Sartory, 1926). The first report of this disease in gorgonians dates back to 1995 [14,15], although similar symptoms and out- breaks had been previously reported in the 1980s [16]. The ascomycete fungus A. sydowii is globally distributed and occurs in diverse environments where it survives as a soil decompos- ing saprotroph [17–19]. It is apparently a terrestrial fungus, but it is salt tolerant and capable of growing in the sea [20]. Moreover, A. sydowii has been reported as a food contaminant [21], and a human pathogen in immune-compromised patients [22,23]. In marine ecosystems, A. sydowii has been isolated from some gorgonian communities of the Caribbean [11,24], Colom- bian Pacific coasts [25], and environmental samples of the Australian coastal waters [20]. Aspergillosis causes selective mortality of large sea fans [26], and suppression of reproduction in infected individuals [27]. As a consequence, coral population levels decrease [28]. The symptoms include purpling of the tissue, galling, and lesions [11], associated with necrotic sea fan tissue [14]. Prevalence (percentage of fans infected) and disease severity (mean percentage of fan tissue affected by disease) are positively correlated with water depth, and large sea fans are more likely to be infected than small fans [15,29]. Although the origin of this disease and its epidemiology is unknown, microsatellites and phylogenetic studies reveal a pattern of global panmixia among isolates. Moreover, sea isolates are interspersed with those isolated from environmental samples [30]. Aspergillus sydowii was isolated, identified and inoculated as the causative agent of the sea fan disease (Koch’s postulates) by previous authors [11,19]. The incidence of this pathogen can be similar to other fungal species, i.e., Fusarium keratoplasticum and F. falciforme, in other animals and ecosystems [31], exacerbated by the effects of global climate change and habitat alteration by humans. In the Ecuadorian Pacific, there are no records of A. sydowii and coral reefs appear to be healthy. Due to the current trend of expansion of fungal infections and the endangered situa- tion of coral reefs, we performed a survey in the Machalilla gorgonian gardens, which includes the most representative gorgonian species in a hot spot of marine biodiversity in Ecuador. We investigated the presence of A. sydowii in these organisms.

Material and Methods Sampling Gorgonian octocoral colonies were collected by SCUBA diving from rocky bottoms located in The Frailes, Machalilla National Park (Manabı´, Ecuador) (1˚30’14"S 80˚48’33”W). The author- ity who issued the permission for each location was the "Ministerio del Ambiente, Manabı´ (ECUADOR)" (Permit Number: N˚ 016 –RM–DPM–MA). Due to the absence of symptoms, we randomly selected 40 colonies from the two most abundant and cosmopolitan gorgonian species of this area (pers. obs.), Leptogorgia Milne-Edwards and Haime, 1857 [32]: Leptogorgia obscura Bielschowsky, 1929 [33], and Leptogorgia sp. (under description). The colonies were collected within a range of 10 to 15 m in depth. Samples were kept in individual sterile plastic bags and processed in the laboratory under axenic conditions.

Fungal isolation From each colony, fragments of ca. 3 cm wide from randomly selected areas were excised using a sterile scalpel. To remove fungi not associated with the octocorals, the selected fragments were surface-sterilized with 70% ethanol for 30 s [25]. For fungal isolations, the selected

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fragments were transferred onto a peptone glucose agar media (PGA) [34] supplemented with penicillin (100 mg/l). In order to avoid any possible errors in the identification of coral fungi (negative control) the sea water sample was isolated. A glass-ring technique was used for isolation following the methodology described in [31]. Resulting pure cultures were maintained in PGA at 4˚C. Cultures were labeled as ASP001 through ASP059 in the culture collection of the Real Jardı´n Bota´nico, Madrid, Spain (Table 1).

DNA extraction, PCR amplification, sequencing, and species identification DNA was extracted from 20 mg of the fungal isolate tissues using the DNeasy extraction kit (Qiagen, Inc.) according to the manufacturer’s protocol. DNA fragments containing internal transcribed spacers ITS1 and ITS2, including 5.8S, were amplified and sequenced with primer

pair ITS5/ITS4 [35]. The PCR profile was: 2.5 μl 10 x buffer, 1.4 μl 50 mM MgCl2, 1.6 μl 25 mM dNTPs, 0.5 μl of each 10 mM primer (forward and reverse), 1 μl 1 mg/ml BSA, 1 μl DNA, 0.3 μl 5 U/μl Taq polymerase, and 16.2 μl ddH2O. The PCR conditions were 1 min at 95˚C, 35 cycles of 1 min at 95˚C, 45 s at 58˚C and 1 min at 72˚C, and finally 10 min at 72˚C. The ampli- cons were sequenced for both strands using BigDye Terminator in an ABI 3730 genetic ana- lyzer (Applied Biosystems). The sequences were edited and primers trimmed using the Sequencher v.4.9 program (Gene Code Corporation, Ann Arbor, MI, USA). BLAST [36] was used to compare the sequences against those existing in the National Center of Biotechnology Information (NCBI) nucleotide databases. For species identification of the isolates, the corresponding ITS sequences were phylogenetically analyzed with a number of selected ITS sequences of reference of closely related fungal species obtained from the NCBI (see Table 2, Fig 1). To perform the phylogenetic analyses, a GTR + G + I substitution model was first obtained using the jModelTest v2.1.5 [37] program. This model was selected based on the Akaike Information Criterion (AIC). Bayesian inference and Maximum Likelihood analyses were performed using MrBayes v3.2.5 [38] and RaxML v8.0.0 [39], respectively. The Bayesian inference analysis was implemented with three runs of 20 million generations sampling one tree per 1000 replicates. For each run, eight Markov chain Monte Carlo (MCMC) simulations were conducted. These simulations were run until a critical value was reached for the topological convergence diagnostic lower than 0.005. Branch supports were evaluated by posterior probabilities after a burn-in of 25%. The Maximum Like- lihood analysis was implemented with a random starting tree and clade support was assessed with 1000 bootstrap replicates. The Maximum Likelihood analysis was implemented in the graphical user interface raxmlGUI v1.5 [40]. The clade support was assessed with 1000 bootstrap replicates after selecting the best tree from 100 trees generated.

Morphological characterization Isolates corresponding to A. sydowii according to phylogenetic analysis were morphologically characterized using scanning electron microscopy, SEM (Hitachi s3000N, Real Jardı´n Bota´- nico, CSIC, Madrid, Spain). Mycelia with characteristic features were fixed in 2% glutaralde- hyde for 1 h, washed in distilled sterile water, and then dehydrated for 1 h in a series of ethanol (30, 50, 70, 80, 90, 95 and 100%) solutions. The isolates dehydrated in absolute ethanol were critical-point dried and the material was sputter coated in a vacuum with an electrically con- ductive layer of gold to a thickness of about 80 nm. Samples were observed at a beam specimen angle of 45˚ with an accelerating voltage of 20kV and final aperture at 200 μm.

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Table 1. Fungal isolates from gorgonians Leptogorgia obscura and Leptogorgia sp. from the Eastern Pacific of Ecuador and the resulting molecular identification based on phylogenetic analysis. RJB number Isolate number Gorgoniidae Species Fungus Species GenBank Acc. Num. ASP001 GORG01 Leptogorgia sp. Pyrenochaetopsis leptospora KX712403 ASP002 GORG02 Leptogorgia sp. Nigrospora sp. KX712404 ASP003 GORG03 Leptogorgia sp. Penicillium chrysogenum KX712405 ASP004 GORG04 Leptogorgia sp. Penicillium chrysogenum KX712406 ASP005 GORG05 Leptogorgia sp. Penicillium chrysogenum KX712407 ASP006 GORG07 Leptogorgia obscura Tritirachium sp. KX712408 ASP007 GORG08 Leptogorgia sp. Penicillium chrysogenum KX712409 ASP008 GORG09 Leptogorgia obscura Penicillium chrysogenum KX712410 ASP009 GORG10 Leptogorgia sp. Penicillium chrysogenum KX712411 ASP010 GORG11 Leptogorgia obscura Penicillium chrysogenum KX712412 ASP011 GORG12 Leptogorgia obscura Fusarium longipes KX712413 ASP012 GORG13 Leptogorgia sp. Penicillium chrysogenum KX712414 ASP013 GORG16 Leptogorgia sp. Nigrospora sp. KX712415 ASP014 GORG17 Leptogorgia obscura Lasiodiplodia pseudotheobromae KX712416 ASP015 GORG18 Leptogorgia obscura Phoma sp. KX712417 ASP016 GORG21 Leptogorgia obscura Penicillium chrysogenum KX712418 ASP017 GORG22 Leptogorgia sp. Penicillium chrysogenum KX712419 ASP018 GORG24 Leptogorgia obscura Fusarium longipes KX712420 ASP019 GORG25 Leptogorgia obscura Cladosporium dominicanum KX712421 ASP020 GORG26 Leptogorgia obscura Aspergillus sydowii KX712422 ASP021 GORG27 Leptogorgia obscura Aspergillus sydowii KX712423 ASP022 GORG29 Leptogorgia obscura Penicillium chrysogenum KX712424 ASP023 GORG30 Leptogorgia sp. Penicillium chrysogenum KX712425 ASP024 GORG31 Leptogorgia obscura Nigrospora sp. KX712426 ASP025 GORG32 Leptogorgia sp. Aspergillus wentii KX712427 ASP026 GORG33 Leptogorgia sp. Penicillium chrysogenum KX712428 ASP027 GORG34 Leptogorgia sp. Penicillium chrysogenum KX712429 ASP028 GORG35 Leptogorgia obscura Penicillium chrysogenum KX712430 ASP029 GORG36 Leptogorgia sp. Penicillium chrysogenum KX712431 ASP030 GORG37 Leptogorgia sp. Penicillium chrysogenum KX712432 ASP031 GORG38 Leptogorgia sp. Cladosporium sphaerospermum KX712433 ASP032 GORG40 Leptogorgia obscura Nigrospora sp. KX712434 ASP033 GORG42 Leptogorgia obscura Fusarium longipes KX712435 ASP034 GORG43 Leptogorgia obscura Penicillium chrysogenum KX712436 ASP035 GORG44 Leptogorgia obscura Penicillium chrysogenum KX712437 ASP036 GORG45 Leptogorgia sp. Penicillium chrysogenum KX712438 ASP037 GORG46 Leptogorgia obscura Penicillium chrysogenum KX712439 ASP038 GORG47 Leptogorgia sp. Penicillium chrysogenum KX712440 ASP039 GORG48 Leptogorgia obscura Nigrospora sp. KX712441 ASP040 GORG49 Leptogorgia obscura Fusarium longipes KX712442 ASP041 GORG50 Leptogorgia sp. Penicillium chrysogenum KX712443 ASP042 GORG51 Leptogorgia sp. Penicillium chrysogenum KX712444 ASP043 GORG53 Leptogorgia sp. Aspergillus ochraceopetaliformis KX712445 ASP044 GORG54 Leptogorgia obscura Nigrospora sp. KX712446 ASP045 GORG55 Leptogorgia obscura Penicillium chrysogenum KX712447 ASP046 GORG63 Leptogorgia sp. Cladosporium sphaerospermum KX712448 (Continued)

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Table 1. (Continued)

RJB number Isolate number Gorgoniidae Species Fungus Species GenBank Acc. Num. ASP047 GORG68 Leptogorgia sp. Penicillium chrysogenum KX712449 ASP048 GORG71 Leptogorgia obscura Capnobotryella sp. KX712450 ASP049 GORG76 Leptogorgia sp. Cladosporium sphaerospermum KX712451 ASP050 GORG77 Leptogorgia sp. Cladosporium sphaerospermum KX712452 ASP051 GORG78 Leptogorgia sp. Curvularia sp. KX712453 ASP052 GORG80 Leptogorgia sp. Alternaria sp. KX712454 ASP053 GORG83 Leptogorgia obscura Aspergillus sclerotiorum KX712455 ASP054 GORG84 Leptogorgia obscura Aspergillus sclerotiorum KX712456 ASP055 GORG85 Leptogorgia sp. Nigrospora sp. KX712457 ASP056 GORG87 Leptogorgia sp. Cladosporium sphaerospermum KX712458 ASP057 GORG88 Leptogorgia sp. Penicillium mallochii KX712459 ASP058 GORG90 Leptogorgia obscura Cladosporium dominicanum KX712460 ASP059 GORG92 Leptogorgia sp. Tritirachium sp. KX712461 doi:10.1371/journal.pone.0165992.t001

Results Fungal isolation and species identification A total of 59 fungal isolates were obtained and the phylogenetic analyses resulted in 17 phylogenetically supported clusters (Fig 1). The majority of the isolates could be assigned to a species reference sequence (from type material). These species were: Penicillium chrysogenum, P. mallochii, Aspergillus sclerotiorum, A. ochraceopetaliformis, A. sydowii, A. wentii, Lasiodiplodia pseudotheobromae, Cladosporium shpaerospermum, C. dominicanum, Fusarium longipes, and Pyrenochaetopsis leptospora. Five groups of isolates grouped with sequences of references of unknown species: Alternaria sp., Capnobotryella sp. Curvularia sp., Phoma sp., and

Table 2. Genbank rDNA ITS reference sequences for fungal species used in phylogenetic analysis to identify fungal isolates from Ecuadorian gorgonians. Species GenBank number Isolate / strain Type material Penicillium chrysogenum NR_077145 CBS 306.48 yes Aspergillus sclerotiorum NR_131294 NRRL 415 yes Aspergillus ochraceopetaliformis KF384187 FJ120 - Aspergillus sydowii AB267812 CBS 593.65 - Penicillium mallochii NR_111674 DAOM 239917 yes Aspergillus wentii NR_077152 ATCC 1023 yes Lasiodiplodia pseudotheobromae NR_111264 CBS 116459 yes Cladosporium sphaerospermum NR_111222 CBS 193.54 yes Cladosporium dominicanum NR_119603 - yes Nigrospora oryzae HQ607925 ATT291 - Nigrospora sp. KP793234 M116 - Nigrospora sp. JN207335 P39E2 - Fusarium longipes AB820724 IFM 50036 - Tritirachium sp. EU497949 F13 - Alternaria sp. KM507780 311a - Curvularia sp. HE861836 UTHSC:08±2905 - Pyrenochaetopsis leptospora NR_119958 - yes Phoma sp. KF367550 4 BRO-2013 - doi:10.1371/journal.pone.0165992.t002

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Fig 1. Bayesian out-group-rooted cladogram inferred from ITS rRNA gene sequences of fungal isolates from Leptogorgia obscura and Leptogorgia sp. from the Eastern Ecuadorian Pacific. Numbers placed above and below the internodes are, respectively, PP and BS of the Bayesian and Maximum Likelihood analyses. doi:10.1371/journal.pone.0165992.g001

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Fig 2. Pie charts showing isolation frequency of different fungal isolates from each host gorgonian species, Leptogorgia obscura and Leptogorgia sp. of the Eastern Ecuadorian Pacific. doi:10.1371/journal.pone.0165992.g002

Tritirachium sp. The sequences grouping into the cluster containing Nigrospora spp. were diverse and could not be assigned to any known ITS sequences (Table 2). The majority of the isolates belonged to the genera Penicillium and Aspergillus (Fig 2). The most frequent species was Penicillium chrysogenum (24 out of 59), which occurred in colonies of both L. obscura and Leptogorgia sp. (Fig 2), followed by Nigorspora spp. and Penicillium granulatum (both 4 out of 59 each), to Aspergillus sydowii (2 out of 59). The species A. sydowii and Fusarium longipes were only found in L. obscura (Fig 2).

Morphological characterization The isolates molecularly assigned to A. sydowii showed conidiophores with conidia, metulae, and phialides seen in the SEM micrographs (Fig 3). The length of the metulae ranged from 2.5 to 3.5 μm, and the breadth from 4.2 to 6.5 μm. Phialides ranged from 2.2 to 3.0 μm in length and from 3.4 to 6.1μm in breadth. The conidia were globose with a roughened or spinose ornamentation. These conidia had a diameter that ranged from 2.5 to 4.4 μm (Fig 3).

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Fig 3. Scanning electron microscopy showing characteristic features of conidiophores of Aspergillus sydowii isolated from Leptogorgia obscura and Leptogorgia sp. of the Eastern Ecuadorian Pacific: (A) conidiophore structure with metule (m), phialide (p), and globose conidia (c); (B) morphological features of conidia head with a vesicle characteristic (v), metulae (m), phialide (p), and globose conidia (c); (C) phialide (p) and mature globose conidia (c) with verruculose ornamentation (*); (D) globose conidia (c) with verruculose ornamentation (*). doi:10.1371/journal.pone.0165992.g003

Discussion The Ecuadorian Pacific coral reefs are one of the most important and unstudied marine ecosystems and biodiversity ‘hot spots’. In this study, we found that the gorgonian communities of these reefs, specifically L. obscura and Leptogorgia sp. colonies, hold a large, diverse, and mostly unknown fungal community in these hosts. Interestingly, this fungal community includes the coral pathogen A. sydowii. This constitutes the first report of this pathogen in Ecuador and the second in the Eastern Pacific. The gorgonian community appeared to be healthy and showed no symptoms of fungal disease during the sampling period. These results are similar to those of [13,41], who also found isolates of A. sydowii in healthy colonies of Gor- gonia ventalina in the Caribbean. In Ecuador, this pathogen was only found in the samples of L. obscura but not in those of Leptogorgia sp. This could be due to the limited sampling and the low prevalence of this fungus in these populations (only 2 specimens out of 59). A different susceptibility of the gorgonian species could also explain this result. A previous study [14] indicated differences in the incidence of aspergillosis between two species of gorgonians, e.g., G. ventalina and G. flabellum.

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Moreover, we provide new data on fungal communities of marine environments, particu- larly in coral reefs. Studies on fungal communities in coral reefs are scarce. In Singapore, an investigation on 10 species of gorgonian corals indicated the presence of 16 fungal genera, including Acremonium, Aspergillus, Chaetophoma, Cladosporium and Penicillium [42]; among these genera, the species Cladosporium sphaerospermum and Phoma sp. were also found in our study. In a study carried out in colonies of the Caribbean sea fan G. ventalina [43], 15 new fungal species were found, corresponding to 8 genera, including Aspergillus, Cladosporium, Gloeo- tinia and Penicillium. In 2008, as part of a larger sampling effort, the same authors identified 35 fungal species corresponding to 15 genera, including Aspergillus, Cladosporium, Nectria, Penicillium and Stachybotrys [13]. Among these genera, the species A. sydowii and C. sphaerospermum were also found in our study. In the Pacific, the only sampled coral reef area studied for fungal community composition was at Choco´, Colombia [25]. This area is located near the coral reef investigated in our study. They found 59 fungal species in the sea fan Pacifigorgia spp. corresponding to 13 fungal genera (e.g., Aspergillus, Penicillium). In our work, we only found A. sydowii and A. sclerotiorum, two species already reported in the eastern Pacific [25]. Thus, all other fungal species identified in our study represent the first report of this species in the Pacific gorgonians. We also found species that had been previously reported in gorgonians. These included Penicillium chrysogenum [13,25,44] and Cladosporium sphaerospermum [43]. However, the role of pathogens of these species remains unknown. We found some fungal species that had never been described in gorgonians, including Aspergillus fumigatus (previously described in soil, air, water, food, plants and organic matter), Capnobotryella sp. (previously described in lichens and pumpkins), Fusarium longipes (previously described in soil of tropical regions), Lasiodiplodia theobromae (previously described causing damage in vascular plants), Phoma sp. (described from soil, as saprophytes on various plants, and as pathogens in plants and humans) and Pyrenochaetopsis leptospora (soil borne and mainly associated with gramineous plants). The pathogenicity of these fungal species to gorgonians is unknown. However, whether these species are also characteristic of marine environments or originate from land as agricultural runoff and move to marine environments via discharges from rivers is unknown. Other species found in this work have been previously found in marine environments but not in gorgonians or coral reefs. For example, Aspergillus ochraceopetaliformis and Alternaria sp. were found in deep-sea environments [45,46], Nigrospora sp. in sea anemones [47], or saline environments in general, such as Penicillium mallochii was before isolated from the guts of tropical leaf-eating caterpillars in Costa Rica [48]. In spite of the presence of known pathogens, such as A. sydowii, and a wide diversity of fungal pathogens in the Ecuadorian coral reefs, we did not observe any damage or mortality. The factors that lead to aspergillosis or the development of other fungal pathogens on coral reefs are unknown. In phylogenetically related fungal pathogens, some environmental stressors influence the development of disease [31,49]. It was suggested that coral aspergillosis could be enhanced by abiotic factors [50,51] and speculated that this disease could be the result of specific combinations of environmental factors (e.g., humidity, UV, temperature, aerosol concentrations) or of large-scale climate patterns (oceanic currents) [50]. Specifi- cally, the microclimatic parameter of temperature has been shown to be involved in the gorgonia-A. sydowii interaction by promoting the growth and activity of the pathogen and reducing the efficacy of host defenses [24,25]. Thus far, studies on coral diseases, such as Aspergillosis, are limited by the lack of isolates from marine sources prior to epidemics [30]. The detection of these pathogens in healthy Ecuadorian gorgonian gardens is, therefore, of key importance since it indicates the presence

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of A. sydowii and other potential coral pathogens in asymptomatic colonies. This can help in further investigations aiming to decipher the factors leading to gorgonian disease and an even- tual deterioration of this ecosystem. Alert models on diverse environmental stressors and the monitoring of the health of coral reefs are crucial for a better understanding of the development of aspergillosis disease. Future studies on this pathogen and the factors triggering disease require the comparison of healthy coral reefs with the presence of A. sydowii to those affected by this pathogen. Understanding where and to what extent potentially harmful organisms exist is of crucial importance to the design of adequate conservation plans. Fungal diseases currently represent one of the main threats to biodiversity worldwide [52] and corals are no exception. The results presented here contribute to a better understanding of the biodiversity of fungal communities in coral reefs and the construction of data-baselines for the presence and incidence of these fungal pathogens in natural systems and in particular coral reefs.

Acknowledgments We thank the Museo Ecuatoriano de Ciencias Naturales, Machalilla National Park and Minis- terio del Ambiente of Ecuador (Manabı´) for participation and collection permits (N˚ 016 – RM–DPM–MA). Thanks to Michel Guerrero and his team (Exploramar Diving) for his special interest from the early stages of our research. Special thanks to Micaela Peña for unconditional help and support. This research was partially supported by a grant from the Spanish Ministry of Economy and Competitiveness (CTM2014-57949-R).

Author Contributions Conceptualization: MMSH JDU. Formal analysis: MMSH JVSS JDU AM. Funding acquisition: AM. Methodology: MMSH JDU JVSS AM. Writing – original draft: MMSH JDU JVSS AM. Writing – review & editing: MMSH JDU JVSS AM.

References 1. Enochs I, Hockensmith G. Effects of coral mortality on the community composition of cryptic metazoans associated with Pocillopora damicornis. Proc 11th Int Coral Reef Symp. 2008; 26: 1368±1372. Avail- able: http://www.nova.edu/ncri/11icrs/proceedings/files/m26-08.pdf 2. Sanchez J, Dueñas LF. Diversidad y evolucioÂn de octocorales. HipoÂtesis, apuntes cientõÂficos uniandi- nos. 2012; 12: 42±46. 3. Buhl M, Mortensen P. Symbiosis in Deep-Water Corals. Symbiosis. 2004; 37: 33±61. 4. Thrush SF, Dayton PK. Disturbance to marine benthic habitats by trawling and dredging: implications for marine biodiversity. Annu Rev Ecol Syst. 2002; 33: 449±473. doi: 10.1146/annurev.ecolsys.33. 010802.150515 5. Tsounis G, Rossi S, Gili JM, Arntz W. Population structure of an exploited benthic cnidarian: the case study of red coral (Corallium rubrum L.). Mar Biol. 2006; 149: 1059±1070. doi: 10.1007/s00227-006- 0302-8 6. Gates RD, Ainsworth TD. The nature and taxonomic composition of coral symbiomes as drivers of per- formance limits in scleractinian corals. J Exp Mar Bio Ecol. Elsevier B.V.; 2011; 408: 94±101. doi: 10. 1016/j.jembe.2011.07.029 7. NystroÈm M, Folke C, Moberg F. Coral reef disturbance and resilience in a human-dominated environ- ment. Trends Ecol Evol. 2000; 15: 413±417. doi: 10.1016/S0169-5347(00)01948-0 PMID: 10998519

PLOS ONE | DOI:10.1371/journal.pone.0165992 November 30, 2016 10 / 12 A. sydowii Gorgonians Ecuador

8. Wilkinson C. Status of coral reefs of the World: 2008. Status Coral Reefs World 2008. 2008; 5±19. 9. Szmant AM. Nutrient enrichment on coral reefs: Is it a major cause of coral reef decline? Estuaries. Coastal and Estuarine Research Federation; 2002; 25: 743±766. Available: http://www.jstor.org/stable/ 1353030 10. Contreras AC, OrtegoÂn-Aznar I, Mota AT, SuaÂrez-Salazar J. Cambio de fase coral-algas en el arrecife de coral de Mahahual, en el Caribe Mexicano. 64th Gulf Caribb Fish Inst. 2011; 28±31. Available: http:// nsgl.gso.uri.edu/flsgp/flsgpw11001/papers/010.pdf 11. Smith GW, Ives LD, Nagelkerken IA, Ritchle KB. Caribbean sea-fan mortalities. Nature. 1996. p. 487. doi: 10.1038/383487a0 12. SaÂnchez JA, GoÂmez CE, Escobar D, Dueñas LF. Diversidad, abundancia y amenazas de los octocorales de la Isla Malpelo, PacõÂfico Oriental Tropical, Colombia. Bol Invest Mar Cost. 2011; 40: 139±154. 13. Toledo-HernaÂndez C, Zuluaga-Montero A, Bones-GonzaÂlez A, RodrõÂguez JA, Sabat AM, Bayman P. Fungi in healthy and diseased sea fans (Gorgonia ventalina): is Aspergillus sydowii always the pathogen? Coral Reefs. 2008; 27: 707±714. doi: 10.1007/s00338-008-0387-2 14. Nagelkerken I, Buchan K, Smith GW, Bonair K, Bush P, GarzoÂn-Farreira J, et al. Widespread disease in caribbean sea fans I. Spreading and general characteristics. In: Lessios HA, Macintyre IG, editors. Proceedings of the 8th International Coral Reef Symposium Vol 1. Smithsonia. Panama; 1997. pp. 679±682. 15. Nagelkerken I, Buchan K, Smith GW, Bonair K, Bush P, GarzoÂn-Ferreira J, et al. Widespread disease in Caribbean sea fans: II. Patterns of infection and tissue loss. Mar Ecol Prog Ser. 1997; 160: 255±263. doi: 10.3354/meps160255 16. GarzoÂn-Ferreira J, Zea S. A mass mortality of Gorgonia ventalina (Cnidaria: Gorgonidae) in the Santa Marta area, Caribbean coast of Colombia. Bull Mar Sci. 1992; 50: 522±526. 17. Klinch MA. Identification of common Aspergillus species. Centraalbureau voor Schimmelcultures. Utrecht, Netherlands.; 2002. 18. Raper KB, Fenell DI. The genus Aspergillus. In: Williams, Wilkins, editors. Baltimore, USA; 1965. 19. Geiser DM, Taylor JW, Ritchie KB, Smith GW. Cause of sea fan death in the West Indies. Nature. 1998; 394: 137±138. doi: 10.1038/28079 20. Hallegraeff G, Coman F, Davies C, Hayashi A, McLeod D, Slotwinski A, et al. Australian dust storm associated with extensive Aspergillus sydowii fungal ªBloomº in coastal waters. Appl Environ Microbiol. 2014; 80: 3315±3320. doi: 10.1128/AEM.04118-13 PMID: 24657868 21. Zhang XY, Zhang Y, Xu XY, Qi SH. Diverse deep-sea fungi from the south china sea and their antimi- crobial activity. Curr Microbiol. 2013; 67: 525±530. doi: 10.1007/s00284-013-0394-6 PMID: 23736224 22. de Hoog GS, Guarro J, GeneÂ J, Figueras MJ. Atlas of clinical fungi. 2nd ed. Utrecht/Reus: Centraal- bureau voor Schimmelcultures/Universitat Rovira i Virgili.; 2000. 23. Nagarajan C, Thayanidhi P, Kindo AJ, Ramaraj V, Mohanty S, Arunachalam R. Fungal Rhinosinusitis: Report of uncommon Aspergillus species as etiological agents. Int J Case Reports Images. 2014; 5: 13. doi: 10.5348/ijcri-2014-01-430-CS-3 24. Alker AP, Smith GW, Kim K. Characterization of Aspergillus sydowii (Thom et Church), a fungal pathogen of Caribbean sea fan corals. Hydrobiologia. 2001; 460: 105±111. doi: 10.1023/A:1013145524136 25. Barrero-Canosa J, Dueñas LF, SaÂnchez JA. Isolation of potential fungal pathogens in gorgonian corals at the Tropical Eastern Pacific. Coral Reefs. 2013; 32: 35±41. doi: 10.1007/s00338-012-0972-2 26. Kim K, Harvell CD. The rise and fall of six a six-year coral-fungal epizootic. Am Nat. 2004; 164: S52± S63. doi: 10.1086/424609 PMID: 15540141 27. Petes LE, Harvell CD, Peters EC, Webb MAH, Mullen KM. Pathogens compromise reproduction and induce melanization in Caribbean sea fans. Mar Ecol Prog Ser. 2003; 264: 167±171. doi: 10.3354/ meps264167 28. Rypien K. The origins and spread of Aspergillus sydowii, an opportunistic pathogen of caribbean gorgonian corals. Faculty of the Graduate School of Cornell University. 2008. 29. Kim K, Harvell CD. Aspergillosis of sea fan corals: disease dynamics in the Florida Keys. In: Porter JW, Porter K, editors. The Everglades, Florida Bay, and coral reefs of the Florida Keys: an Ecosystem Sourcebook. CRC Press. New York; 2002. pp. 813±824. 30. Rypien KL, Andras JP, Harvell CD. Globally panmictic population structure in the opportunistic fungal pathogen Aspergillus sydowii. Mol Ecol. 2008; 17: 4068±4078. doi: 10.1111/j.1365-294X.2008.03894.x PMID: 18684135 31. Sarmiento-Ramirez JM, Abella-Perez E, Phillott AD, Sim J, Van West P, Martin MP, et al. Global distribution of two fungal pathogens threatening endangered sea turtles. PLoS One. 2014; 9. doi: 10.1371/ journal.pone.0085853 PMID: 24465748

PLOS ONE | DOI:10.1371/journal.pone.0165992 November 30, 2016 11 / 12 A. sydowii Gorgonians Ecuador

32. Milne-Edwards H, Haime J. Histoire naturelle des coralliaires ou, Polypes proprement dits. Paris: à la Libraire EncyclopeÂdique de Roret; 1857. http://dx.doi.org/10.5962/bhl.title.11574 33. Bielschowsky E. Die Gorgonarien Westindien. 6. Die Familie Gorgoniidae, zugleich eine Revision. Zool JahrbuÈcher, Suppl: . 1929; 16: 63±234. 34. SoÈderhaÈll K, Svensson E, Unestam T. Chitinase and protease activities in germinating zoospore cysts of a parasitic fungus, Aphanomyces astaci, Oomycetes. Mycopathologia. 1978; 64: 9±11. doi: 10.1007/ BF00443081 35. White TJ, Bruns S, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A guide to methods and applications. 1990. pp. 315±322. citeulike- article-id:671166 36. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990; 215: 403±10. doi: 10.1016/S0022-2836(05)80360-2 PMID: 2231712 37. Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and high-per- formance computing. Nat Methods. 2012; 9: 772. doi: 10.1038/nmeth.2109 PMID: 22847109 38. Huelsenbeck JP, Ronquist F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics. 2001; 17: 754±755. doi: 10.1093/bioinformatics/17.8.754 PMID: 11524383 39. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. Oxford University Press; 2014; 30: 1312±1313. doi: 10.1093/bioinformatics/btu033 PMID: 24451623 40. Silvestro D, Michalak I. RaxmlGUI: a graphical front-end for RAxML. Org Divers Evol. 2012; 12: 335± 337. doi: 10.1007/s13127-011-0056-0 41. Zuluaga-Montero A, Toledo-HernaÂndez C, RodrõÂguez JA, Sabat AM, Bayman P. Spatial variation in fungal communities isolated from healthy and diseased sea fans Gorgonia ventalina and seawater. Aquat Biol. 2009; 8: 151±160. doi: 10.3354/ab00218 42. Koh LL, Tan TK, Chou LM, Goh NKC. Fungi associated with gorgonians in Singapore. In: Moosa MK, Soemodihardjo S, Soegiarto A, Romimohtarto K, Nontji A, Suharsono S, editors. Proceedings of the Ninth International Coral Reef Symposium. 2000. pp. 521±526. 43. Toledo-HernaÂndez C, Bones-GonzaÂlez A, Ortiz-VaÂzquez OE, Sabat AM, Bayman P. Fungi in the sea fan Gorgonia ventalina: diversity and sampling strategies. Coral Reefs. 2007; 26: 725±730. doi: 10. 1007/s00338-007-0252-8 44. Moree WJ, McConnell OJ, Nguyen DD, Sanchez LM, Yang Y-L, Zhao X, et al. Microbiota of healthy corals are active against fungi in a light-dependent manner. ACS Chem Biol. American Chemical Society; 2014; 9: 2300±2308. doi: 10.1021/cb500432j PMID: 25058318 45. Zhang X, Tang G, Xu X, Nong X, Qi S-H. Insights into deep-sea sediment fungal communities from the east indian ocean using targeted environmental sequencing combined with traditional cultivation. Cha- turvedi V, editor. PLoS One. 2014; 9: e109118. doi: 10.1371/journal.pone.0109118 PMID: 25272044 46. Landy ET, Jones GM. What is the fungal diversity of marine ecosystems in Europe? Mycologist. 2006; 20: 15±21. doi: 10.1016/j.mycol.2005.11.010 47. Yang K-L, Wei M-Y, Shao C-L, Fu X-M, Guo Z-Y, Xu R-F, et al. Antibacterial anthraquinone derivatives from a sea anemone-derived fungus Nigrospora sp. J Nat Prod. American Chemical Society; 2012; 75: 935±941. doi: 10.1021/np300103w PMID: 22545792 48. Rivera KG, DõÂaz J, ChavarrõÂa-DõÂaz F, Garcia M, Urb M, Thorn RG, et al. Penicillium mallochii and P. guanacastense, two new species isolated from Costa Rican caterpillars. Mycotaxon. 2012; 119: 315± 328. doi: 10.5248/119.315 49. DieÂguez-Uribeondo J, FoÈrster H, Adaskaveg JE. Effect of wetness duration and temperature on the development of anthracnose on selected almond tissues and comparison of cultivar susceptibility. Phy- topathology. 2011; 101: 1013±20. doi: 10.1094/PHYTO-07-10-0193 PMID: 21521000 50. Green EP, Bruckner AW. The significance of coral disease epizootiology for coral reef conservation. Biol Conserv. 2000; 96: 347±361. doi: 10.1016/S0006-3207(00)00073-2 51. Rypien KL. African dust is an unlikely source of Aspergillus sydowii, the causative agent of sea fan disease. Mar Ecol Prog Ser. 2008; 367: 125±131. doi: 10.3354/meps07600 52. Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, et al. Emerging fungal threats to animal, plant and ecosystem health. Nature. 2012; 484: 186±194. doi: 10.1038/nature10947 PMID: 22498624

PLOS ONE | DOI:10.1371/journal.pone.0165992 November 30, 2016 12 / 12